Equine sperm refers to the male reproductive cells produced by stallions, essential for the process of fertilization and successful breeding in horses. The study of equine sperm encompasses various aspects, including morphology, motility, viability, and genetic integrity. These parameters are critical for assessing stallion fertility and improving breeding outcomes. Research in this field often focuses on understanding the factors that influence sperm quality, such as age, nutrition, and environmental conditions. Additionally, advancements in assisted reproductive technologies, such as artificial insemination and cryopreservation, rely heavily on the detailed study of sperm characteristics. This page compiles peer-reviewed research studies and scholarly articles that explore the biology, evaluation, and technological applications related to equine sperm.
Setchell BP, Duggan MC, Evans RW.Single intravenous injections of ovine luteinizing hormone (LH) in adult hamsters and rats had no effect on fluid secretion by the testes, as measured by the gain in weight or water content during a 10-h period after ligation of the efferent ducts (EDL). Neither was there any obvious effect on the liberation of spermatozoa, as judged by the total number of sperm in the unligated and EDL testes and from the concentration of spermatozoa in the secreted fluid, calculated from the difference between the number of sperm in the EDL and unligated testes divided by the difference in weight.
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Brooks DE.1. A method is described for the extraction, purification and separation of acid-soluble phosphorus compounds from mammalian semen. [8-(14)C]ATP and [8-(14)C]AMP were used as internal recovery standards to measure the breakdown and loss of these nucleotides in the procedure. 2. Bull, ram, boar and stallion semen was separated into seminal plasma and spermatozoa and the two fractions were examined separately. The overall composition of the mixture of the phosphorus compounds extracted from the two fractions was similar for the four species. 3. Glycerylphosphorylcholine and glycerylphosphorylino...
The effects of cryopreservation on the acrosomal status of equine spermatozoa were investigated. Ejaculates (n=10) from six stallions were processed fresh, after cooled storage at 4-6 degrees C for 24 h in either a milk-based or lactose-EDTA freezing extender and after freeze-thawing in lactose-EDTA extender in liquid nitrogen at either 5 x 10(7) or 2 x 10(8) spermatozoa ml(-1). All samples were incubated in TALP-TEST for 2 h at 39 degrees C in 5% CO2. Subsamples were challenged with calcium ionophore A23187 for 10 min. The acrosomal status of the spermatozoa was evaluated by staining the sper...
Mares were inseminated with motile spermatozoa suspended in 30-150 microliters Tyrode's medium directly onto the uterotubal papilla at the anterior tip of the uterine horn, ipsilateral to the ovary containing a dominant preovulatory follicle of > or = 35 mm in diameter, by means of a fine gamete intrafallopian transfer (GIFT) catheter passed through the working channel of a strobed light videoendoscope. Insemination of 10, 8, 25, 14, 11 and 10 mares with, respectively, 10.0, 5.0, 1.0, 0.5, 0.1 or 0.001 x 10(6) motile spermatozoa resulted in conception rates of, respectively, 60, 75, 64, 29,...
The objective of this study was to enhance the in vitro sperm quality and in vivo fertility of frozen-thawed equine semen by the addition of l-carnitine (LC) to post-thawed semen. Different concentrations of LC were added to thawed samples to obtain four treatments control and 0.5, 1 and 2 mM LC. In the in vitro experiments, sperm motility and kinematics, membrane integrity and intracellular calcium ion concentration ([Ca ] ) were investigated, and the antioxidant bioactivity of LC was assessed by measuring hydrogen peroxide and nitrite concentrations (NO ). The fertility rate was assessed v...
Maria Feitosa Ribeiro L, Bersot Gomes JV, Hernando Ortiz Vega W, da Silva Alves A, Netto Machado M, Junio Wutke L, Raquel Quirino C....Stallion breeding is mostly based on desirable phenotypic traits, with little consideration for semen quality. Objective: To identify relationships among semen parameters in stallions using a non-invasive, integrative approach across summer and winter seasons. Methods: Twenty-four stallions were evaluated, in which semen collections, testicular thermography, and Doppler velocimetry of the testicular artery by Doppler ultrasonography were performed, in addition to the measurement of physiological, environmental, and heat stress parameters, both in winter (coat weather) and summer (T-shirt weath...
Ramírez-Agámez L, Hernández-Avilés C, Samper JC, Love CC.Intracytoplasmic sperm injection (ICSI) using frozen/thawed (F/T) stallion sperm is a common procedure in the equine breeding industry. Historically, sex-sorted (SS) F/T stallion sperm has yielded lower cleavage (<30 %) and blastocyst rates (<5 %) after ICSI when compared to non-sorted (NS) F/T sperm. Recently, a new technology for sperm sex-sorting (Genesis III) has been validated by a commercial company. In Experiment 1, the post-thaw quality between NS-F/T and SS-F/T stallion sperm produced with this technology was compared. The post-thaw sperm motility was higher in NS-F/T (41 %) than i...
Brito LFC, Linardi RL, Rosales LAS, Balamurugan NS, Hernández-Avilés C, Ramírez-Agámez L.Efficient use of stallion semen in liquid state is limited by its relatively short shelf-life. A chemically defined extender (Beyond) is now available for long-term liquid semen preservation. The objectives of the present study were to compare Beyond with milk extenders for the preservation of semen at two temperatures, and to evaluate fertility of semen cooled for 4-8 days before artificial insemination. Semen was processed using different extenders: milk, cholesterol (BotuSemen Special); milk-based (INRA 96); and Beyond. Sperm motility, membrane and acrosome integrity, and chromatin struct...
de Zutter BM, de Paula Freitas-Dell'Aqua C, Dell'Aqua-Junior JA, Monteiro GA, Troncarelli T, Papa FO.This study evaluated the effects of cholesterol, pentoxifylline and casein, with or without skim milk, added to extenders during pre-centrifugation on equine sperm cryosurvival. Seven ejaculates from four stallions (n = 28) were divided into four groups: SM (skim milk), SMP (SM + pentoxifylline), SMCho (SM + cholesterol) and ChoCa (cholesterol + casein). After centrifugation and freezing, sperm kinematics and plasma membrane integrity were assessed immediately and 30 min post-thaw. SMCho and ChoCa showed superior results compared with SM and SMP. These findings indicate that chol...
Solanki O, Talluri TR, Jhamb D, Kumar S, Pargi KK, Paul N, Kutty M, Dedar RK, Meena M, Gaur M, Bhattacharya TK.The current study evaluated the effect of L-Carnitine supplementation to the stallion semen extender on pre-freeze and post-thaw semen quality and oxidative parameters in Marwari breed stallions. Methods: For this purpose, thirty-six semen ejaculates (6 ejaculates from each stallion) were collected using an artificial vagina (AV) from six adult Marwari stallions. The gel free semen was extended with different concentrations of L-Carnitine (LC) i.e. 0 mM (control), 2.5 mM (T1), 5 mM (T2) and 10 mM (T3). Pre-freeze and post-thaw semen evaluations were conducted for various seminal attributes and...
Broothaers K, Jouneau A, Angel-Velez D, De Coster T, Festuccia N, Archilla C, Calderari S, Jouneau L, Van den Branden E, Peere S, Polfliet E....In vitro production of equine embryos has been performed using intracytoplasmic sperm injection (ICSI) for the last two decades. Since 2022, a repeatable protocol for conventional in vitro fertilization (IVF) provides a successful alternative. However, little is known about the influence of the fertilization method on embryo quality and the transcriptomic profile. In this study, we aimed to examine differentially expressed genes (DEGs) between ICSI and IVF embryos in the horse. Therefore, ten equine sibling blastocysts, produced in vitro by either ICSI or IVF from three different mares, were s...
Martin-Pelaez S, Orsolini M, Verstraete M, de la Fuente A, Takahashi K, Meyers S, Dini P.Equine in vitro embryo production (IVP) offers a valuable opportunity to enhance the efficient utilization of genetically and economically valuable horses. In cases of subfertility, sudden death, emergency euthanasia, or limited sperm availability, IVP becomes the only viable option to produce offspring. Due to steadily improving success rates, IVP has become one of the most sought-after assisted reproductive techniques. The rising global demand has created a clear need for veterinarians and stallion owners to provide IVP-suitable sperm, where the selection of intact sperm for intracytoplasmic...
Otero JC, Neild DM, Ferrante AA, Gambarotta MC, Caldevilla ML.Glycerol, a penetrating cryoprotectant, is most employed for deep freezing spermatozoa. However, it can induce toxic, chemical, and osmotic effects, altering the lipid structure of the sperm membrane. Rapid glycerol addition has been demonstrated to have fewer toxic effects than its removal. Objective: To minimize both the toxic and osmotic effects of glycerol on equine sperm through post-thaw dilution using two different extenders. Methods: Samples of equine semen frozen with 5% glycerol from nine stallions were thawed, re-diluted 1:2, and incubated for 30 minutes at 22°C in Tyrodes (Tyr) an...
Jones CJP, Aplin JD, Wilsher S.There is little information on non-ciliated secretory cell numbers in the equine oviduct isthmus over the course of the estrous cycle and the effect, if any, of insemination. Lectin histochemistry was used on a series of 13 oviducts taken at different stages of the estrous cycle, with and without insemination, to monitor glycosylation changes and the distribution of secretory cells characterised by a prominent cytoplasmic accumulation of glycoconjugates. There appeared to be an increase in secretory cells expressing a wide range of glycans in all specimens at estrus. One day after ovulation, o...
Bueno VLC, Bastos HBA, Centeno LAM, Schmitt F, Larentis G, Mattos RC, Fiala-Rechsteiner S.The seminal plasma (SP), a fluid originating from the testes, epididymis, and accessory sex glands, represents 98 % of the volume of stallion ejaculate. Metabolomic analysis enables the identification of final products of metabolic pathways, providing insights into reproductive physiology. Objective: This study aimed to investigate the metabolomic profile of stallion seminal plasma and to investigate metabolic differences between stallions with high and low pregnancy rates, in order to identify potential biomarkers associated with fertility. Methods: Twenty-four Criollo stallions, were sampled...
Khalil WA, Sharf MI, Derbala MK, Hassan MAE, Abdelnour SA, El-Harairy MA.This study aims to investigate the cryoprotective effect of Omega-3 nano-emulsion (Omega-3 NE) on stallion sperm quality, kinematic parameters, acrosome status, subcellular ultrastructure, oxidative/antioxidant markers, and semen microbiota. Forty ejaculates were collected, extended, and cryopreserved from 5 fertile Pure Egyptian stallions (Equus caballus). The ejaculates were divided into five groups: a control group (without additive) and four groups supplemented with 25, 50, 100, and 200 μg of Omega-3 NE/mL. The Omega-3 NE exhibited an average particle size of 51-146 nm, a PDI of 0.58,...
Rychtarova J, Fulka H, Loi P, Fulka J.In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and i...
Rodrigues MF, Neves AP, Ferreira R, Pessoa GA, Araujo DN, de Souza GV.Melatonin has demonstrated beneficial effects in regulating inflammatory processes in various tissues, but its role in mare uterine physiology remains unclear. Objective: To investigate the effects of melatonin on the gene expression of cytokines (IL-1β, IL-6, IL-8, IL-10, and TNF-α) in equine uterine explants cultured for 6, 12, or 24 hours. Methods: The study was divided into two experiments. Part 1 aimed to evaluate the cytotoxicity of melatonin at concentrations of 0, 0.5, 1, 2, 4, and 8 mM on endometrial cells. Part 2 aimed to determine the expression of inflammatory cytokines in uterin...
Latorre N, Gómez-Cuétara C, Cañizares E, Crespo F, Pérez-Aguilera V, Cuerda MI, Laborda-Gomariz JA, Soler AJ, Roldan ERS, Sanchez-Rodriguez A.Evaluating semen quality in Purebred Spanish Horses is essential to determine reproductive potential and optimize assisted reproductive techniques. Objective: This study compared several sperm analysis methods in Purebred Spanish Horses to standardize protocols suitable for both laboratory and field conditions. Methods: Fifteen stallions were evaluated through three ejaculates, analyzing sperm parameters using different techniques. Sperm kinematics was assessed by Sperm Computer Analyzer (SCA-CASA) or iSperm mCASA. Viability was analyzed by eosin-nigrosin-Giemsa (ENG) staining or Hoechst 33258...
Becerro-Rey L, Martín-Cano FE, Silva-Rodríguez A, Ortega-Ferrusola C, da Silva-Álvarez E, Zabalo-Palomo C, Gil C, Peña FJ.Commercial extenders for equine semen typically contain high glucose concentrations; however, recent research indicates that these supraphysiological concentrations of glucose may be detrimental. Spermatozoa lack translational activity and depend on post-translational protein modifications for their regulation. Supraphysiological glucose concentrations are known to modify cell regulation, and we hypothesize that post-translational modification deregulation associated with supraphysiological glucose concentrations occurs in spermatozoa extended in high glucose media; and increasing the concentr...
Pinto-Bravo P, Rebordão MR, Amaral AS, Szóstek-Mioduchowska A, Fernandes C, Galvão AM, Silva E, Alpoim-Moreira J, da Costa RPR, Skarzynski DJ....Oviduct glycoprotein 1 (OVGP1) is a key protein involved in oviductal functions. β-estradiol (E) and progesterone (P), oxytocin (OXT) and tumor necrosis factor-α (TNFα) modulate the equine oviduct function, through prostaglandin regulation. The objective was to evaluate OVGP1 expression within each equine oviduct segment (infundibulum, ampulla isthmus), throughout the estrous cycle. The in vitro effect of (i) E, P, OXT, TNFα; and (ii) spermatozoa, on oviduct OVGP1 transcription and secretion was studied. Gene transcription was assessed by real-time PCR; protein expression by western blot; ...
Dindo S, Tovar-Pascual L, Baldassarro VA, Bucci D, Mislei B, Spinaci M, Ortiz-Rodriguez JM.Nanoplastics (NP) accumulation in biological tissues and their adverse effects on fertility through inflammatory and oxidative stress responses have recently been described as consequences of global plastic pollution. However, little is known about the impact of NP on gametes. This study aimed to assess the internalization of NP and their effects on mature equine spermatozoa. Frozen-thawed ejaculates from five stallions were divided into untreated control (CTR) and samples supplemented with different concentrations (10, 50, 100 and 200 μg/mL) of 30 nm polystyrene NP. At baseline (T0), and a...
Larriva-González F, Erráez-Guaicha JE, Torres-Ordóñez KE, Duma M, Larriva-Salazar S, Galarza DA.This study evaluated the cryoresistance of stallion sperm frozen by ultra-rapid (UR) methods using microspheres and straws or by the conventionally-slow (CS) method. Sixteen ejaculates from four stallions were each divided into three aliquots according to the freezing method: UR freezing in 30-μL spheres (UR-Spheres) by direct immersion in liquid nitrogen (LN); UR freezing in 0.25-mL straws (UR-Straws) by direct horizontal submersion in LN; and CS freezing in LN vapors. Ultra-rapid freezing medium included 100 mM trehalose +1 % BSA, and the CS freezing medium contained 5 % dimethylformamid...
Hernández-Avilés C, Varner DD, Ramírez-Agámez L, Samper JC, Love CC.In the current study, we report the effect of different commercially available semen freezing extenders utilized for the "test-freeze" procedure for 13 years (2010-2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility - TM [%]; progressive motility - PM [%]; and curvilinear velocity - VCL [μm...
Khorsand F, Hamali H, Qasemi-Panahi B, Tohidkia M.This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane ...
Griffin RA, Miller K, Colyvas K, Sheridan A, De Iuliis G, Aitken RJ, Baker MA, Gibb Z, Swegen A.Exposure to systemic heat stress in male mammals adversely affects sperm production, fertility, and DNA integrity. To date, few studies have investigated this phenomenon in horses, particularly in industry-relevant environments. Therefore, this study examined the relationship between ambient climatic conditions and fertility within a population of commercially fertile stallions. Post-coital semen samples ( = 804) were collected weekly from 46 Thoroughbred stallions during two successive breeding seasons (NSW, Australia; 22 weeks total). Semen samples were processed via single-layer colloid...
Becerro-Rey L, Martín-Cano FE, Silva-Rodríguez A, Ortega-Ferrusola C, da Silva-Álvarez E, Ortiz-Placín C, Tapia JA, Gil MC, Peña FJ.Three isoforms of lactate dehydrogenase (LDH) - LDHA (cytoplasmic), LDHB (mitochondrial), and LDHC (flagellar) - have been identified and localized in stallion spermatozoa. Functional inhibition assays indicate that these three isoforms constitute a lactate shuttle of crucial importance for sperm function. Unassigned: Stallion spermatozoa use different energy sources; while oxidative phosphorylation predominates, glycolysis and beta-oxidation of fatty acids are also present. Glycolysis depends on the availability of NAD+ as an electron acceptor. During glycolysis, NAD+ is reduced to NADH. To e...
